权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Direct observation of dynein-microtubule binding

直接观察动力蛋白-微管结合

基本信息

批准号：
15370064
负责人：
KAMIYA Ritsu
金额：
$ 5.82万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15370064/
关键词：
Cilia and flagella Dynein arm Microtubule Total reflection microscope Fluorescence label Tektin

项目摘要

In thee outer doublet microtubule in the flagellar axoneme, the outer dynein arms are arranged with a 24 nm interval, while each of the seven kinds of inner arms are arranged with a 96 nm interval. How such a regular arrangement is produced is almost entirely unknown. In this study, we aimed to approach this mechanism by directly observing the dynein-microtubule binding process. Using total reflection optics and fluorescently labeled proteins, we observed that the association of dynein to the microtubule is a cooperative process, quantified the cooperative binding using outer-dynein-arm-docking complex (ODA-DC) expressed in insect culture cells. Along with these studies, we searched for proteins responsible for the binding of inner dynein arms and discovered that the protein tektin is involved in the attachment of dynein e in Chlamydomonas axonemes. This is the first study to show tektin involvement in the dynein arrangement ; tektin, first discovered in sea urchin sperm flagella, has been thought to function as a ruler that determines the longitudinal periodicity in the axoneme. In a still different line of experiment, we examined the dynamic nature of dynein-microtubule association using a technique called fluorescence recovery after photobleaching. Using electroporation-mediated incorporation of fluorescently labeled dynein subunit, we found that inner arm dynein of Chlamydomonas is constantly being turned over within the flagella. These findings should provide important insights into the mechanism as to how dynein arms are attached to axonemal microtubules.

鞭毛轴突的三种外双线微管中，动力蛋白外臂以24 nm的间隔排列，7种内臂各以96 nm的间隔排列。这种规律的安排是如何产生的几乎完全不为人所知。在本研究中，我们旨在通过直接观察动力蛋白与微管的结合过程来探讨这一机制。利用全反射光学和荧光标记蛋白，我们观察到动力蛋白与微管的结合是一个协同过程，并利用昆虫培养细胞中表达的外动力蛋白臂对接复合物（ODA-DC）定量了这种协同结合。在这些研究中，我们寻找了负责内动力蛋白臂结合的蛋白质，发现蛋白质tektin参与了衣藻轴突体中动力蛋白e的附着。这是第一个显示tektin参与动力蛋白排列的研究；Tektin最早是在海胆精子鞭毛中发现的，它被认为是决定轴突纵向周期性的尺子。在另一个不同的实验中，我们使用一种称为光漂白后荧光恢复的技术检查了动力蛋白-微管结合的动态性质。利用电穿孔介导的荧光标记动力蛋白亚基的掺入，我们发现衣藻的内臂动力蛋白在鞭毛内不断地被翻转。这些发现应该为动力蛋白臂如何附着在轴突微管上的机制提供重要的见解。